AmpC β-lactamase enzymes have disseminated worldwide on plasmids since the late 1980s and now represent a substantial clinical threat. Their presence renders the bacteria resistant to most β-lactams including cephamycins and β-lactam/β-lactamase combinations, which are generally stable against extended-spectrum β-lactamases (ESBLs). There are currently no standard diagnostic tests available for detection of plasmid mediated AmpC (pAmpC).Objectives: Detection and genetic identification of molecular types of pAmpC in clinical isolates of Echerichia coli, Klebsiella spp. and Proteus mirabilis that lack chromosomal cephalosporinase. Comparing different phenotypic tests to the PCR as a gold standard test to determine a simple method for detection of pAmpC-producers in the clinical laboratory. Methods: Sixty of 178 Enterobacterial isolates were included according to selection criteria: resistance to third generation cephalosporins, to cephamycins and absence of chromosomal cephalosporinase. Bacterial species were identified using API E20 (BioMerieux, France). AmpC genes clusters were amplified by PCR followed by DNA sequencing of the amplified PCR products. AmpC-producers were phenotypically detected using cloxacillin and aminophenyl boronic acid (APB)-based tests as well as AmpC disk test. Clonal relatedness in ampC-positive Klebsiella isolates was assessed using PFGE. ESBL-producers were detected using modified double disk synergy test (MDDST). Susceptibility of ampC positive isolates to cefipime, ciprofloxacin, amikacin and imipenem was detected. Results: AmpC genes were detected in 28.3% (17/60), ESBLs in 53.3% (32/60) of test isolates and 10 isolates produced both ESBLs and pAmpC enzymes (16.7%). CMY-2 was the most prevalent AmpC enzyme produced (70.4%), followed by DHA-1 (23%) then CMY-4 (5%). Cloxacillin-based tests showed better sensitivity (82%) in detecting AmpC enzymes than the APB-based tests and the AmpC disk test (76%). PFGE showed no clonal relatedness between ampC-positive isolates. All studied isolates remained sensitive to imipenem. Conclusion: This study reveals the high prevalence of pAmpC and ESBL enzymes among bacterial isolates from our hospital. It announces the detection of CMY-2, DHA-1 and CMY-4 of the AmpC enzymes in our region. Cloxacillin disk test (200µg/ml) is a feasible and reliable method that accurately detects the majority of ampC-positive isolates particularly in ESBL-positive isolates (sensitivity 70%, specificity 100%). Imipenem remains the best treatment option in treating serious infections caused by AmpC-producing isolates even in case of co-production of ESBL enzymes.